A colleague extracted RNA from chicken brain for me to perform qPCR targeting 4 specific genes and using 18S as the housekeeping gene. He did trizol extraction. When I nanodropped his samples the 230/260 ratios were very low, indicating possible chloroform/phenol contamination. We decided to run the samples through columns from an extraction kit to try to clean them. The ratios were closer to 2 and the 260/280 were also good. I took the samples to our core facility to get the concentrations on a Qubit machine.
From there I made cDNA using iScript kit. I started testing the primers, the sequences we got from previously published papers. The 18S primers had a perfect efficiency of about 100. However, all the other primer sets were giving identical Ct values at all five concentrations of cDNA I was testing (1:10 to 1:100K), or didn't work at all. We ran the melting curve to look for dimers but the melting curves looked normal I've never had anything like this happen on previous qPCR projects. I'm completely flummoxed.
Based on the success with the 18S gene, you can rule out issues with the qPCR machine, cDNA template, qPCR mix, and other shared reagents.
The issue might just be the primers for the 4 other genes.
Are these newly designed primers? Have you tried them before? Did you get any amplification of the expected-size products?
Try and get each set of primers working individually (e.g. set up a small number of reactions with a full set of controls, run part of the reaction out on an agarose gel to check the size).
Good luck!
From my point of view and your result, maybe there is a problem with your Tm temperature of your primer. In my opinion, try a gradient PCR for your primer to find precise Tm of your primers.
Good luck.
A few basic checks (which you may already have done): what do you get if you use the primers with no cDNA at all?
If you still see the same Cq and a melt peak, then...contaminated primers: buy more (or make up fresh stocks).
If I get data that isn't consistent with dilution, it's usually either contaminated primers or primer dimers.
You could also try seeing what you get with cDNA prepared with no reverse transcriptase (-RT control): this will identify if you're amplifying genomic DNA at all.
It's also probably worth looking at your raw Cq values and asking yourself whether they accord with your expectations: you can also relate this to your 18S data. I would expect 18S to be ridiculously abundant, so Cq values of 9-12 with ~5ng cDNA. If you're getting 18S Cq values that are markedly higher, then it could be that you just don't have much cDNA (poor synthesis or other issues): low overall cDNA will affect all your analysis, and make all the above sources of potential issues much more likely to occur.
Similarly, if you're finding your GOIs are giving you Cq values of 28+, consider that they might not be great primers, or your target isn't meaningfully there.
Even though these are primers from the literature, it might be worth validating them yourself: do you have positive controls you can use to confirm/deny what you're seeing?
Also also, while I think of it: you said 260/230 ratios were low, but what were the yields? If the yields were good but the ratios poor, that's fixable with columns (or reprecipitation), because the issue there is contamination. If the yields were poor, then the ratios will also be poor even with minimal salt contamination (because it's a ratio). Here column purification will mostly just lose more of your already low-yield RNA. The fact you had to quantify via qubit tends to suggest this latter is more likely: for basic, straightforward belt & braces qPCR, I would expect the starting material to be meaningfully quantifiable by nanodrop (i.e. at least 50-100ng.ul-1, ideally >200ng.ul-1).
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